A method for evaluating the host range of bacteriophages using phages fluorescently labeled with 5-ethynyl-2′-deoxyuridine (EdU)
A method for evaluating the host range of bacteriophages using phages fluorescently labeled with 5-ethynyl-2′-deoxyuridine (EdU)
复制标题
使用 5-乙炔基-2-脱氧尿苷 (EdU) 荧光标记的噬菌体评估噬菌体宿主范围的方法
DOI：
10.1007/s00253-012-4174-1
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发表时间：
2012
影响因子：
5
通讯作者：
Sayaka Ohno
中科院分区：
文献类型：
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作者：
小原良和;高橋恒二;神納健太郎;山中一司;Yusuke Takahashi; Takato Mitsudome; Tomoo Mizugaki; Koichiro Jitsukawa; Kiyotomi Kaneda;御纏真美子，平山道世，高山大鑑，岩瀬顕秀，工藤昭彦，根岸雄一;Sayaka Ohno
噬菌体(噬菌体)宿主范围的评估是理解噬菌体和原核生物群落相互作用的一个重要问题。然而，在传统的方法，如空斑试验，目标宿主菌株必须分离，尽管几乎所有的环境原核生物是顽固的培养。在这里，我们介绍了一种新的利用荧光标记噬菌体评估噬菌体宿主范围的方法(FLP方法)，该方法包括以下四个步骤：(I)将荧光标记噬菌体加入到微生物联合体中，感染宿主细胞并进行荧光标记。(Ii)荧光细胞通过荧光激活细胞分选进行分选。(3)对从分选细胞中提取的16S rRNA基因序列进行了分析，并设计了荧光原位杂交(FISH)特异的寡核苷酸探针。(4)用荧光标记的噬菌体和FISH探针标记的细胞被鉴定为宿主细胞。为了验证该方法的可行性，我们以T4噬菌体和大肠杆菌为模型。我们首先使用了核酸染色试剂来标记噬菌体；然而，这些试剂也对非宿主细胞进行了染色。接下来，我们使用了Invitgen公司的Click-it Edu(5-乙炔基-2‘-脱氧尿苷)检测试剂盒进行噬菌体标记。利用EDU标记的T4噬菌体，我们可以特异性地检测到E。来自城市污水的复杂微生物联合体中的大肠菌群。我们还证实了鱼可以应用于受感染的E。结肠癌细胞。这些结果表明，这种使用EDU试剂盒的FLP方法是评估噬菌体宿主范围的一种有用的方法，并可能对各种类型的噬菌体具有潜在的应用价值，即使它们的原核宿主目前是不可培养的。
The evaluation of bacteriophage (phage) host range is a significant issue in understanding phage and prokaryotic community interactions. However, in conventional methods, such as plaque assay, target host strains must be isolated, although almost all environmental prokaryotes are recalcitrant to cultivation. Here, we introduce a novel phage host range evaluation method using fluorescently labeled phages (the FLP method), which consists of the following four steps: (i) Fluorescently labeled phages are added to a microbial consortium, and host cells are infected and fluorescently labeled. (ii) Fluorescent cells are sorted by fluorescence-activated cell sorting. (iii) 16S rRNA gene sequences retrieved from sorted cells are analyzed, and specific oligonucleotide probes for fluorescence in situ hybridization (FISH) are designed. (iv) Cells labeled with both fluorescently labeled phage and FISH probe are identified as host cells. To verify the feasibility of this method, we used T4 phage andEscherichia colias a model. We first used nucleic acid stain reagents for phage labeling; however, the reagents also stained non-host cells. Next, we employed the Click-iT EdU (5-ethynyl-2′-deoxyuridine) assay kit from Invitrogen for phage labeling. Using EdU-labeled T4 phage, we could specifically detectE. colicells in a complex microbial consortium from municipal sewage. We also confirmed that FISH could be applied to the infectedE. colicells. These results suggest that this FLP method using the EdU assay kit is a useful method for evaluating phage host range and may have a potential application for various types of phages, even if their prokaryotic hosts are currently unculturable.